Expression Of Fluorescent Reporter Protein Research Articles

Activation of cardiac fibroblast phenotypes following myocardial ischemia‐reperfusion injury in transgenic mouse models

Following myocardial injury, cardiac fibroblasts are believed to play important roles in inflammatory and wound healing processes. We hypothesized that activation of cardiac fibroblasts and myofibroblasts in response to ischemia‐reperfusion (I/R) injury could be detected with transgenic mouse strains expressing fibroblast phenotype‐sensitive promoters linked to expression of fluorescent reporter proteins (Collagen I promoter:Enhanced Green Fluorescent Protein, Coll I:EGFP or Smooth Muscle α‐Actin promoter:Red Fluorescent Protein, SMA:RFP). Activation of cardiac fibroblasts was monitored by reporter fluorescence in frozen tissue sections and by FACS analysis of enzymatically dispersed fibroblast populations. Frozen sections from hearts of reporter mice sacrificed 4 days after I/R injury induced by transient coronary artery (LAD) occlusion showed robust enhancement in cells expressing CollI:EGFP and modest increases in SMA:RFP fluorescence. Fibroblast activation was localized to the infarct zone, and was not observed in control non‐injured mice. FACS analysis of cardiac fibroblasts isolated after I/R injury showed elevations of fluorescent reporter expression (EGFP>RFP). The respective fibroblast phenotypes were maintained under culture conditions. In conclusion, fibroblast phenotype‐sensitive reporters show activation of fibroblasts in the injured myocardium in vivo.

Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing

Optical emission computed tomography (optical-ECT) is a technique for imaging the three-dimensional (3D) distribution of fluorescent probes in biological tissue specimens with high contrast and spatial resolution. In optical-ECT, functional information can be imaged by (i) systemic application of functional labels (e.g. fluorophore labelled proteins) and/or (ii) endogenous expression of fluorescent reporter proteins (e.g. red fluorescent protein (RFP), green fluorescent protein (GFP)) in vivo. An essential prerequisite for optical-ECT is optical clearing, a procedure where tissue specimens are made transparent to light by sequential perfusion with fixing, dehydrating and clearing agents. In this study, we investigate clearing protocols involving a selection of common fixing (4% buffered paraformaldehyde (PFA), methanol and ethanol), dehydrating (methanol and ethanol) and clearing agents (methyl salicylate and benzyl-alcohol-benzyl-benzoate (BABB)) in order to determine a 'fluorescence friendly' clearing procedure. Cell culture experiments were employed to optimize the sequence of chemical treatments that best preserve fluorescence. Texas red (TxRed), fluorescein isothiocyanate (FITC), RFP and GFP were tested as fluorophores and fluorescent reporter proteins of interest. Fluorescent and control cells were imaged on a microscope using a DSred2 and FITC filter set. The most promising clearing protocols of cell culture experiments were applied to whole xenograft tumour specimens, to test their effectiveness in large unsectioned samples. Fluorescence of TxRed/FITC fluorophores was not found to be significantly affected by any of the test clearing protocols. RFP and GFP fluorescence, however, was found to be significantly greater when cell fixation was in ethanol. Fixation in either PFA or methanol resulted in diminished fluorescence. After ethanol fixation, the RFP and GFP fluorescence proved remarkably robust to subsequent exposure to either methyl salicylate or BABB. The optimized optical clearing procedure of ethanol fixation followed by methyl salicylate clearing preserved the fluorescence of constitutive RFP in whole xenograft tumour specimens, about 1 cc in dimension, indicating successful extension from cell plating experiments to whole tissue samples. Finally, the feasibility of imaging the 3D distribution of viable tumour cells (as indicated by the RFP emission) is demonstrated by optical-ECT imaging of cleared xenograft tumours using an in-house system.For more information on this article, see medicalphysicsweb.org